Monoclonal antibody recognizes a conformational epitope in a random coil protein

The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not recognize intact native cytochrome c, but they crossreact with the heme-containing peptides 1-38 and 1-65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2-4 is conformational and discontiguous, it is composed of residues close to the N-terminus and around position 25. The other monoclonal antibody, Cyt-1-59, seems to recognize a contiguous epitope close to the N-terminus. The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme-catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2-4 is particularly interesting because this antibody inhibits the proliferation of a T-cell clone specific for apo-cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547-548].     

Dipeptide backbone conformation and antibody recognition of a viral octapeptide epitope.

The peptide YKGTMDSG (Tyr-Lys-Gly-Thr-Met-Asp-Ser-Gly) represents an important antigenic determinant from the glycoprotein G2 of the pathogenic Rift Valley fever virus. By preparing a series of single-residue substitution peptides, the importance to antigenicity of individual residues within this octapeptide has been determined. Here, we investigated a simple and rapid computational analysis to test for correlations between the observed antigenicity of the substitution analogue peptides and the calculated conformational preferences in local regions of the peptides. Conformational energy analyses were carried out on all dipeptide combinations represented in the wild-type octapeptide and in the single-residue substitution analogue peptides. Conformational similarities and differences between wild-type and substitution dipeptide pairs were determined. The results of these computational analyses were then compared with the data on the relative antigenicity of the wild-type octapeptide and the substitution analogues. This comparison revealed a positive correlation. Substitution peptides showing changes in antigenicity possessed significant changes in the calculated backbone conformation relative to wild type in the dipeptides encompassing the residue substitution. Substitution peptides showing no change in antigenicity similarly showed no significant changes in dipeptide conformation. The potential utility of dipeptide conformational energy analyses and this preliminary structure-activity correlation are discussed.     

Monoclonal antibody (VII-M31) to bovine factor VII: a specific epitope in the gamma-carboxyglutamic acid domain.

A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.     

Monoclonal antibody ID5: epitope characterization and minimal requirements for the recognition of polyglutamylated alpha- and beta-tubulin

A monoclonal antibody (ID5) raised against the synthetic tetradecapeptide corresponding to the C-terminal region of detyrosinated alpha-tubulin showed an unexpected cross-reactivity with beta-tubulin from pig brain tissue. The specificity and the minimal epitope requirements of ID5 were characterized by competitive enzyme-linked immunosorbent assay (ELISA) and spot blots using a series of synthetic peptides and the natural peptides of beta-tubulin and detyrosinated alpha-tubulin from brain. The epitope of ID5 is comprised of the carboxyterminal sequence -XEE carrying the terminal alpha-carboxylate group with X being a variable residue. All linkages in the epitope involve alpha-peptide bonds. This epitope is provided by the detyrosinated alpha-tubulin main chain and the polyglutamyl side chains of both brain alpha- and beta-tubulins. Affinity purification of beta-tubulin peptides and mass spectrometric characterization reveal that peptides carrying three to nine glutamyl residues in the side chain are recognized by ID5. These results show that except for the first gamma-peptide linkage the alpha-peptide bond is the preferred linkage type in the tubulin polyglutamyl side chains.     

Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody.

The role of renal expression of the glycosphingolipid verotoxin receptor, globotriaosylceramide, in susceptibility to verotoxin-induced hemolytic uremic syndrome is unclear. We show that a single glycosphingolipid can discriminate multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide binding profile for verotoxin 1, verotoxin 2 and monoclonal anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact alpha-galactose and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome. For most samples, verotoxin 1 immunostaining within adult glomeruli was observed (type A). Some samples, however, lacked glomerular binding (type B). Anti-globotriaosylceramide (and less effectively, verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B glomerular/tubular globotriaosylceramide showed minor, but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following pretreatment with cold acetone, or methyl-beta-cyclodextrin, used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric, but no adult glomerular staining; this was confirmed by anti-fluorescein isothiocyanate immunostaining. Acetone induced fluorescein isothiocyanate-verotoxin 1B glomerular staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native verotoxin 1B deoxyglobotriaosylceramide analogue binding showed an alteration in subspecificity. These studies indicate a marked heterogeneity of globotriaosylceramide expression within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same glycosphingolipid. Verotoxin 1 derivatization can induce subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular globotriaosylceramide expression may play a significant (cholesterol-dependent?) role in determining renal pathology following verotoxemia.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver

We previously described a cell surface reactive monoclonal antibody, MAb OC.10, which recognizes an epitope shared by rat fetal liver ductal cells, hepatic progenitor cells, mature cholangiocytes, and hepatocellular carcinomas (HCC). Here, intrasplenic injection of MAb OC.10 into newborn rats was shown by immunofluorescence microscopy to strongly label intrahepatic bile ducts. Furthermore, the in situ labeling of intrahepatic cholangiocytes by injecting MAb OC.10 increased the number of intraportal and intralobular bile ducts with well-defined lumens when compared to IgM-injected control animals. The antigen for MAb OC.10 was identified by mass spectrometry as Hsc70, a constitutively expressed heat shock protein belonging to the HSP70 family. Immunoblot analysis demonstrated that MAb OC.10 reacted with recombinant bovine Hsc70 protein, with protein immunoprecipitated from rat bile duct epithelial (BDE) cell lysates with monoclonal anti-Hsc70 antibody, and with Hsc70-FLAG protein over-expressed in human 293T cells. In addition, Hsc70-specific small interfering RNA reduced the amount of OC.10 antigen expressed in nucleofected BDE cells. Consistent with the specificity of MAb OC.10 for Hsc70, heat shock did not induce OC.10 expression in BDE cells, a characteristic of Hsp70. Immunofluorescence with BDE cells further suggested that MAb OC.10 binds a novel cell surface epitope of Hsc70. This was in contrast to a commercially available monoclonal anti-Hsc70 antibody that showed strong cytosolic reactivity. These findings demonstrate that presentation of the OC.10 epitope differs between cytosolic and surface forms of Hsc70 and may suggest distinct differences in protein conformation or epitope availability determined in part by protein–protein or protein–lipid interactions. Phage display and pepscan analysis mapped the epitope for MAb OC.10 to the N-terminal 340–384 amino acids of the ATPase domain of rat Hsc70. These findings suggest that MAb OC.10 recognizes an epitope on rat Hsc70 when presented on the cell surface that promotes morphogenic maturation of bile ducts in newborn rat liver. Furthermore, since we have shown previously that the OC.10 antigen is expressed on HCC subpopulations with oval cell characteristics, our current results indicate that Hsc70 has the potential to be expressed on the surface of certain tumor cells.     

Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart

Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in na?ve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.     

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.     

Substrate specificity of the beta-lactamases found in a number of clinical strains of gram-negative bacteria

The substrate profiles of beta-lactamases defected in 46 clinical polyresistant strains of gram-negative bacteria were determined. By the substrate profile and sensitivity to inhibitors (dicloxacillin and p-CMB) beta-lactamases were considered to belong to classes I, II, III, IV and V of the Richmond classification. The molecular weights of the enzymes were measured. Enterobacter aerogenes 6803, Enterobacter aerogenes 11030 and Klebsiella pneumoniae 970 produced simultaneously two beta-lactamases belonging to different classes. beta-Lactamases of classes I and III were detected in the cells of Enterobacter aerogenes 6803. The cells of Enterobacter aerogenes 11030 contained beta-lactamases of classes V and III and the cells of Klebsiella pneumonia 970 beta-lactamases of classes II and III. Therefore, in all the cases one of beta-lactamases belonged to the class III enzyme close to TEM beta-lactamases by its substrate profile, molecular weight and sensitivity to the inhibitors. Cephalexin and dicloxacillin were most frequently stable to the effect of the above beta-lactamases. The enzymes from 26 strains did not hydrolyse or hydrolysed slightly cephalexin and the enzymes from 19 strains did not hydrolyse of hydrolysed slightly dicloxacillin.     

The solution structure of the bacterial HSP70 chaperone protein domain DnaK(393-507) in complex with the peptide NRLLLTG

The Hsp70 family of molecular chaperones participates in a number of cellular processes, including binding to nascent polypeptide chains and assistance in protein (re)folding and degradation. We present the solution structure of the substrate binding domain (residues 393-507) of the Escherichia coli Hsp70, DnaK, that is bound to the peptide NRLLLTG and compare it to the crystal structure of DnaK(389-607) bound to the same peptide. The construct discussed here does not contain the alpha-helical domain that characterizes earlier published peptide-bound structures of the Hsp70s. It is established that removing the alpha-helical domain in its entirety does not affect the primary interactions or structure of the DnaK(393-507) in complex with the peptide NRLLLTG. In particular, the arch that protects the substrate-binding cleft is also formed in the absence of the helical lid. 15N-relaxation measurements show that the peptide-bound form of DnaK(393-507) is relatively rigid. As compared to the peptide-free state, the peptide-bound state of the domain shows distinct, widespread, and contiguous differences in structure extending toward areas previously defined as important to the allosteric regulation of the Hsp70 chaperones.     

Monoclonal antibody against a lactose epitope of glycosphingolipids binds to melanoma tumour cells

Mice were immunized with a neoglycoprotein consisting of a chemically modified carbohydrate moiety (reductively aminated 3-sialyllactose) linked to human serum albumin. By this procedure an antibody response to the normally non-immunogenic carbohydrate structure was obtained. Hybridomas were established, and monoclonal antibodies were selected in ELISA based on their binding to the saccharide hapten, or to a lactosylceramide-mimicking neoglycolipid, lactose-bis-sulfone. One of the selected antibodies, 2H4, was of particular interest, since it also bound to glycolipids present on melanoma cells. FACS analysis of a panel of 14 melanoma cell lines showed that the 2H4 antibody bound to the majority of these. In frozen, non-fixed sections or paraffin sections of biopsies the monoclonal antibody 2H4 stained melanoma cells, but not tumour infiltrating lymphocytes or normal skin. Detailed immunochemical analysis of 2H4, using thin layer chromatography revealed that it recognized an internal lactose epitope in several glycosphingolipids.Abbreviations BSA bovine serum albumin - FACS fluorescence activated cell sorter - ELISA enzyme-linked immunosorbent assay - HSA human serum albumin - LacCer lactosylceramide - MAb monoclonal antibody - PBS phosphate buffered saline     

The neuroprotective potential of heat shock protein 70 (HSP70)

In response to many metabolic disturbances and injuries, including stroke, neurodegenerative disease, epilepsy and trauma, the cell mounts a stress response with induction of a variety of proteins, most notably the 70-kDa heat shock protein (HSP70). Whether stress proteins are neuroprotective has been hotly debated, as these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury. A few groups have now shown that overproduction of HSP70 leads to protection in several different models of nervous system injury. This review will cover these studies, along with the potential mechanisms by which HSP70 might mediate cellular protection.     

低浓度苯并[a]芘诱导赤子爱胜蚓HSP70和HSP90转录上调研究

为寻找土壤低浓度多环芳烃污染分子生物标记物,采用了抑制消减双杂交的方法构建了赤子爱胜蚓在苯并[a]芘（BaP）人工土壤污染胁迫下的差异表达cDNA文库,经测序和基因比对分析后,在上调文库中分别发现2个与热休克蛋白HSP70和1个与HSP90显著匹配的cDNA克隆.经定量PCR验证了0。1 mg·kg^-1和1。0 mg·kg^-1 BaP对赤子爱胜蚓HSP70和HSP90的诱导作用,表明这两个新克隆到的赤子爱胜蚓热休克蛋白基因可作为土壤污染监测的备选分子生物标记物.     

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.     

Monoclonal antibody with specificity to a conserved epitope in the C-terminal domain of histone H1 variants.

A monoclonal type M-immunoglobulin (IgM) was generated in mice against a nuclease-urea extract of HeLa metaphase chromosomes. This antibody stains metaphase chromosomes from a variety of mammalian cultured cell types by indirect immunofluorescence. Antibody 12C7 reacts by western transfer technique with histone H1 in all the cell lines tested. The antibody cross-reacts with H1, and H1(0) in human cells. Proteolytic digestions of H1 suggest that the epitope is localized in the carboxy-terminal domain of the histone H1 molecule. Digestion with trypsin demonstrates that the antibody 12C7 does not react with the globular domain of histone H1. The C-terminal domain of H1 subtypes therefore seems to have a conserved determinant which does exist in H1, H1(0), and probably in H5. This antibody has applications in studying the role of that domain of H1 in processes like chromosome condensation and variations in chromatin structure which influence gene expression.     

Heat shock protein 70 (HSP70) in gastric adaptation to aspirin in Helicobacter pylori infection.

We have recently shown that adaptation of gastric mucosa to aspirin (ASA) is disturbed in Helicobacter pylori (H. pylori)-infected human stomach, but can be restored by eradication of the bacterium. The aim of this study was 1) to evaluate the influence of H. pylori on expression of heat shock protein 70 (HSP70) during ASA ingestion in these subjects and in mice model and 2) to evaluate, whether altered HSP70 expression might be associated with different adaptation to ASA in H. pylori-positive and eradicated subjects. The gastric mucosal HSP 70 gene expression was determined by quantitative RT-PCR and Western blot and immunohistochemistry during 14 days of ASA ingestion (1 g bid) in the same 8 subjects before and 3 months after successful eradication of H. pylori. In addition, HSP70 mRNA and protein expression were examined in 30 mice without and with H. pylori infection and eradication. During 14 days of ASA treatment, human H. pylori-infected mucosa revealed a decrease of HSP70 expression, while after eradication a higher expression and further increase of HSP70 expression during ASA ingestion were observed. Mice inoculated with H. pylori also exhibited decreased gastric mucosal HSP70 mRNA expression that was restored after eradication therapy. Decreased basal and ASA-induced expression of HSP70 may partly be responsible for impaired gastric adaptation to ASA in H. pylori-positive subjects. We conclude that 1. The HSP70 gene and protein expression is reduced during infection with H. pylori in men and mice and that gastric adaptation to ASA in H. pylori eradicated subjects is accompanied by increased HSP70 expression; 2. It is reasonable to assume that decreased HSP70 expression might contribute to disturbed gastric adaptation in H. pylori infection in humans and 3. The expression of HSP70 plays an important role in the mechanism of gastric adaptation to ASA and that H. pylori infection interferes with this adaptation due to decrease of HSP70 expression in gastric mucosal cells.     

Localisation of a protein core-specific epitope from gastrointestinal mucin (MUC2). The effect of epitope immobilisation on antibody recognition

Human intestinal mucins are high molecular weight glycoproteins which protect and lubricate the epithelium of the gastrointestinal tract. In cases of malignant disease, mucins are abnormally expressed, overproduced or underglycosylated. This feature may enable the mucins to serve as tumour markers. The MUC2 mucin largely consists of a variable number of tandem repeats of a 23 amino acid sequence, ¹PTTTPITTTTTVTPTPTPTGTQT²³. In this study we have localised the minimal and the optimal epitope within this region by the previously developed protein core specific 996 monoclonal antibody using synthetic peptides. Several overlapping and truncated peptides related to the tandem repeat unit have been prepared by solid-phase methodology. Other mucin peptides were synthesised on the tips of polyethylene pins, and these remained C-terminally attached to the pins for comparative investigations. The interaction of the 996 monoclonal antibody with the synthetic peptides was studied either in solution by competition RIA or on immobilised peptides by indirect ELISA experiments. These experiments show that the minimal epitope recognised by the 996 antibody is the Ac-¹⁹TGTQ²² (IC₅₀=3100 μm in solution). For the optimal 996 antibody binding in solution the ¹⁶PTPTGTQ²² heptapeptide (IC₅₀=3 μm ) is required. © 1998 European Peptide Society and John Wiley & Sons, Ltd.